Bright Outlook for Exosome-Based Therapeutics and Diagnostics Despite Numerous Hurdles

Exosomes hold real promise as alternative drug-delivery vehicles with better targeting capabilities and fewer side effects than current synthetic technologies. With potential to deliver nearly all types of drug substances from viral vectors as gene therapies to multiple forms of RNA, DNA, proteins, and small molecule active pharmaceutical ingredients (APIs), exosomes are creating real excitement as a new therapeutic modality. Their production by most cell types in high concentrations also makes them attractive as biomarkers of disease. However, developers must overcome manufacturing, regulatory, funding, and patient recruitment challenges if the full potential of exosomes is to be realized.

A Little about Exosomes

Given the difficulty in delivering naked genetic material across cell membranes, it should come as no surprise that the human body has developed its own delivery systems. Extracellular vesicles (EVs) are nanoscale particles comprising lipid bilayers containing cellular material that are secreted by many different types of cells. They include larger microvesicles, apoptosomes, and migrasomes, which range from 50 nm to 1 mm and exosomes, at 40–160 nm.1

Exosomes are of particular interest, as they have been shown to play an important role in intercellular communication, numerous physiological processes, and disease pathologies.2 They transport not only lipids but DNA and RNA, proteins, enzymes, carbohydrates, cytokines, membrane transporters, and many other bioactive molecules.2-4 Once they reach their target cells, exosomes deliver these materials, which influence proliferation, differentiation, migration, survival, gene expression, cell metabolism, and other functions IN the recipient cells.3

Most exosomes exhibit specific traits correlating to those of their parent cells and are often designed to communicate with specific cell types. For these reasons, exosomes are being explored as disease biomarkers, natural drug-delivery vehicles for immunotherapies, gene therapies, and other drug substances, and in a few cases as drug targets.1

They can be isolated from body fluids, such as semen, urine, milk, bronchoalveolar lavage fluid, and plasma or purposely produced via in vitro cell culture.5 Most of the latter leverage mesenchymal stem cells (MSC) for exosome expression, including bone marrow, umbilical cord, and adipose-derived MSCs.

Exosomes from stem cells are attracting particular interest because they exhibit similar properties to these cells and thus can be used in tissue regeneration and many other therapeutic applications but are easier to store and transport than live cells and do not present immunogenicity and related concerns.2,4 Researchers are also investigating exosomes from dermal, lung, neural, immune, and other cells to enable targeted delivery of drugs to these specific tissues. Plant-derived exosomes, meanwhile, are finding use in nutraceutical products.3

In general, exosomes intended for use in therapeutic applications are generated via cell culture. In addition to the cell type, the particular cell line must be considered, as different cell lines may have different levels of stability as passage numbers increase, and they may secrete different quantities of exosomes under comparable conditions.6 Engineered cell lines are therefore often used to increase expression levels and ensure stability. Use of immortalized cell lines, while not possible with cell therapies, is a practical approach for exosome generation, as they are more cost-effective and also ensure phenotypic and genotypic stability.

Naïve, or natural, exosomes have been shown to exhibit therapeutic benefits.1 MSC-derived exosomes have regenerative and anti-inflammatory properties, while those from lung spheroid cells can reduce the impact of respiratory diseases, those from neural stem cells have been explored as neuroprotective and regenerative therapies, and exosomes from dendritic immune cells exhibit antitumor immune activity. Engineered exosomes, meanwhile, allow for drug loading, greater stability, improved uptake, and enhancement of other performance attributes.3

Most programs are at the preclinical development stage, but a few have progressed into clinical trials. One market research firm estimated the exosome therapy market to be expanding at a compound annual growth rate of 41.1% from a value of $32 million in 2022.7

Diagnostic Potential

Exosomes have significant potential as disease biomarkers because they are known to play pivotal roles in disease pathways, including tumor metathesis, and are released by many different types of cells, including those that are healthy, unhealthy, or modified in some way.1 Containing many different bioactive molecules, exosomes are also generally highly representative of their parent cells and include information on the entire genome, even for tumor and other damaged cells, and thus afford greater diagnostic accuracy. In addition, their presence at relatively higher concentrations than many other biomarkers in most bodily fluids enables detection using various liquid biopsy methods. Furthermore, exosomes are relatively easy to isolate and concentrate, providing higher sensitivity, and fairly stable under reasonable, low-temperature storage conditions.

For these many reasons, exosomes are being investigated as biomarkers for many different types of diseases. Over half of projects involve the detection of many different types of cancer (e.g., breast, lung, liver, prostate, gastric, colorectal, and different carcinomas).8,9 Other diseases for which exosome-based diagnostics are being developed include neurological diseases, such as Parkinson’s disease and chronic traumatic encephalopathy (CTE), cardiovascular diseases, and lung diseases, such as asthma.9 They are also being investigated for the diagnosis of a variety of pregnancy-related issues.

Therapeutic Opportunities

The therapeutic potential of exosomes largely relates to the fact that these nanoparticles are native to the body.8 Consequently, they are not seen by the immune system as foreign and do not illicit undesirable immune responses.

Exosomes also have potentially higher stability and greater targeting capability than man-made delivery vehicles, allowing for reduced dosages and dosing frequencies. Lipid nanoparticles (LNPs) and many other current carrier technologies, on the other hand, suffer from dose-dependent toxicity and ineffective delivery to target tissues.1

Engineered exosomes can support high drug loading levels for many different types of drug substances, ranging from DNA and RNA to viruses, proteins, antisense oligonucleotides (ASO), many types of RNA molecules, and small molecule APIs.8 For the latter, exosomes also enable enhanced solubility, which is an issue for the majority of chemical drug candidates today. Many exosomes also have the ability to cross the blood–brain and air–blood barriers.1

These many possible advantages are driving exploration of exosomes as delivery vehicles across many indications, including infectious, cardiovascular, neurological, orthopedic, metabolic,8 dermatologic,2 and many other diseases,8 as well as many subspecialties of surgical practice.4 According to the CAS Content Collection™, the areas of greatest focus include cancer, neurological and neurodegenerative diseases, lung diseases, and wound healing.9

The cargos can be loaded after the exosomes have been purified, within cells after exosome expression, or as part of exosomes expressed by cells. In addition, they can be attached to the surface or the interior of the exosome.

Manufacturing Considerations and Challenges

Manufacturing exosomes requires their production, isolation from cellular material, purification, and in some cases drug loading and/or other modifications following purification. One of the main hurdles to bringing exosome-based products to the market is the need to establish truly robust, scalable manufacturing processes.

Culture conditions have a significant impact on exosome production — not just the quantity but the contents contained within the generated nanoparticles. Two-dimensional versus three-dimensional cultures, as well as the type of bioreactor (e.g., stirred-tank, rotating-wall, fixed-bed, hollow-fiber) must be evaluated to determine which approach provides the desired exosomes in the highest yields.1

The biggest challenge, however, relates to purification of exosomes, as they must be separated from the other contents of the cultured cells, including other extracellular vesicles, host-cell DNA, host-cell proteins and peptides, and other cellular debris.1,8 These impurities are present in greater quantities and are much larger than the exosomes. In addition, developing standard methods is difficult, as each type of exosome exhibits physicochemical properties.

Many of these impurities can be difficult to separate from the desired exosomes. Methods that have been used include centrifugation (e.g., ultra, density-gradient, filtration, differential), tangential-flow filtration (TFF), immunoaffinity capture, precipitation, ion-exchange and size-exclusion chromatography, and microfluidic technologies, among others.1,8 The potential for exosome degradation during purification leading to negative impacts on structural integrity and functionality is an added complication.5

Centrifugation methods generally provide low recoveries and are time-consuming and expensive, precluding their use for commercial production. TFF is promising but often must be used in conjunction with other techniques, such as immunocapture or chromatography, to reduce the contents of all impurities to acceptable levels. Microfluidics, meanwhile, has yet to be validated for GMP production. Some commercial kits are available to aid in small-scale separation of exosomes, but they are expensive and cannot ensure optimal results.8

Exosome heterogeneity in terms of both quantity and content poses difficulties for the establishment of industry-wide analytical standards as well. Common techniques employed for exosome identification, characterization, and purity analysis are intended to evaluate their morphology, density, surface markers, and contents. They include nanoparticle tracking analysis, transmission electron microscopy (TEM) and cryo-EM, atomic force microscopy, nano-flow cytometry with fluorescent detection, western blot, enzyme-linked immunosorbent assay (ELISA), protein concentration and identification methods, genetic sequencing, lipid analysis, and so on.

Drug loading for therapeutic applications can be achieved using either passive or active methods or during exosome expression.1,5,7 Passive methods involve incubating the exosomes in the presence of the payload molecule at a specific temperature to enable its diffusion into the interior. While the simplest approach, loading efficiencies are often low, and control of content from one exosome to another is difficult. Active methods include sonication, electroporation, surfactant-enabled permeation, and dialysis. These techniques, however, lead to damage of the exosome membrane and thus are impractical for production of GMP therapeutics.

Exosomes with loaded drug substances can also be generated during cell culture via transfection of plasmids encoded to produce the active substance packaged in the exosomes. It is also possible to load drug substances onto the surfaces or in the interiors of exosomes via in situ assembly and synthesis processes.

Most of these methods suffer from suboptimal loading/expression levels. Greater understanding of exosome generation and release is needed to enable development of more cost-effective and efficient exosome manufacturing and purification processes for both naïve and engineered exosomes without/with loaded cargoes (interior and exterior).

Regulatory Questions

As an emerging modality, exosome-based diagnostics and therapeutics face regulatory uncertainties. The heightened level of interest has, however, attracted the interest of regulatory bodies, including the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA), both of which are drafting guidelines for their manufacture and use.10 The lack of definitive guidance and standardization across agencies poses difficulties. In the meantime, trade groups, such as the International Society for Extracellular Vesicles (ISEV), are supporting developers with clarity on the definition of exosomes in the pharmaceutical context and recommended practices for isolation and purification.

One particular issue with exosomes is their classification. As cellular products that contain genetic material, they can be viewed as traditional biologic products and as gene therapies. The two drug classes are regulated under different guidelines, and the need to meet regulatory requirements for quality and safety for both can be a huge hurdle as well.

Clinical Landscape

In June 2022, approximately 70 clinical trials involving exosomes were reported on various registries.11 About two-thirds involved exosomes derived from MSCs. The most common trials involving therapeutic products target SARS-CoV-2 infections and/or acute respiratory distress syndrome.11

In 2023, a total of 120 exosome-based candidates were in development, with approximately 40% of the candidates under investigation having reached the clinical stage of development, and seven of them progressing to later-stage studies. Overall, just under one-third of candidates targeted some form of cancer.

Aegle Therapeutics, Aruna Bio, Avalon Globocare, Capricor Therapeutics, Codiak Biosciences (now defunct), Direct Biologics, Exopharm Ltd, Organicell Regenerative Medicine, RION, and United Therapeutics are some of the companies that have pursued or are pursuing clinical trials with exosome-based candidates.1,10,12 These companies have used exosomes derived from bone-marrow-derived MSCs, platelets, human amniotic fluid, cardiosphere-derived cells, and exosomes. There are also clinical trials underway using plant-derived exosomes to treat colon cancer, oral mucositis related to neck and head cancer, and insulin resistance and inflammation in ovary syndrome patients.8

In addition to studies involving exosome-based therapeutics, there are several clinical trials exploring the use of exosomes in diagnostic applications. Many are focused on the diagnosis of different types of cancer, including various forms of breast, ovarian, lung, colon, and pancreatic cancers.8

It is interesting to note that some of these therapies leverage the ability of exosomes to be recognized as “self” by the human body to deliver active drug substances known to be effective but to cause unacceptable toxic side effects when delivered systemically. One example is exoIL-12, originally developed by Codiak Biosciences and since acquired by Lonza, which is looking to find a partner to take the candidate further. Recombinant interleukin 12 (IL-12) exhibits excellent antitumor efficacy in preclinical models but results in unacceptable side effects in humans. Loading of IL-12 onto the surface of exosomes allows for potent and prolonged activation of T and natural killer (NK) cells in the tumors without any Grade 3 or higher adverse events.13 Codiak also developed a candidate designed to deliver a STING (cGAS/stimulator of interferon gene) agonist that exhibits 100-fold higher potency than the free agonist with a much better safety profile.

Unfortunately, due to the funding issues facing the biopharmaceutical industry in recent years and challenges recruiting patients, some trials have not yet started, while others have had to be halted.5 In addition, some candidates that have realized positive results in early-phase trials have not yet been taken further. These trials include exosome-based treatments for a variety of indications, from cancer to infectious diseases to autoimmune disorders (e.g., Crohn’s diseases, ulcerative colitis), osteoarthritis, Alzheimer’s disease, depression/anxiety, wound healing, skin diseases, bone tissue defects, dry eyes, diabetes, and polycystic ovary syndrome, among others.

Selected Companies in the Exosome Space

Despite the funding challenges, the potential presented by exosomes continues to entice many biotech and pharmaceutical companies to pursue exosome-based product development. According to one market research report, over 60 companies and academic/research institutes have or are pursuing the development of exosome-based therapeutics and diagnostics.7 They have managed, according to the report, to raise approximately $500 million from strategic investors.

Some companies have focused on developing candidates based on naïve exosomes (with and without loading of drug substances), while others have elected to create engineered exosomes. The former include GS Therapeutics, Avalon, Aegle Therapeutics, ExoCoBio, AgeX Therapeutics, Kimera Labs, and United Therapeutics, while Capricor, Evox, Ilias, Carmine Therapeutics, ReNeuron, Anjarium, Adipomics, Brainstorm Cell Therapeutics, Exocure Biosciences, Empty Cell, ILIAS Biologics, The Cell Factory, Evox Therapeutics, Exopharam, Carnine Therapeutics, and TriArm Therapeutics are representatives of the latter.1,12 A few are exploring both approaches, such as EverZom.

Most of the candidates developed by these companies have not proceeded past the preclinical stage. Many of the companies have focused on establishing platform technologies that can be applied to multiple indications. Other examples of companies that have explored preclinical candidates include VivaZome, Vitti Labs, Xollent Biotech, Exocel Bio, and Florica Therapeutics.9

Mercy Bioanalytics, Bio-Techne, Exosome Sciences, Exosomics, Echo Biotech, the University of Texas MD Anderson Cancer Center, a collaboration between Harvard Medical School, and Wenzhou Medical University (China), and researchers at the University of California, San Francisco Medical Center have been involved in efforts to develop diagnostic technologies leveraging exosomes.1,12

A Few CDMOs of Note

While some large biopharmaceutical companies are investing in the development of exosome-based therapeutics and diagnostics, the majority of candidates under investigation belong to small and emerging biotech firms. A few have been able to attract sufficient funding to support development and implementation of in-house manufacturing solutions, but many must rely on outsourcing partners for many of these activities. That has driven interest in exosome production and purification by contract development and manufacturing organizations (CDMOs).10

Some of these CDMOs are established firms supporting the development and manufacturing of traditional and next-generation biologics, such as Lonza. Others have been involved in providing stem-cell-related products and services, such as RoosterBio.1

Many are new, smaller firms themselves that have gotten involved in supporting developers in what they view as a highly promising field. Some are offering outsourcing services in addition to developing their own exosome-based candidates. ExoXpert, EXO Biologics, and EverZom, for example, claim to have established proprietary upstream and downstream manufacturing solutions.1,10 Others offer specialized media products, research reagents, and analytical technologies, including Clara Biotech, NanoView Biosciences, System Biosciences, and The Cell Factory.1

Advancing Exosome Technologies

As natural packages of genetic and other cellular material, exosomes hold real promise as alternative drug-delivery vehicles with better targeting capabilities and fewer side effects than currently used, synthetic technologies. With potential to deliver nearly all types of drug substances from viral vectors as gene therapies to multiple forms of RNA, DNA, proteins, and small molecule drug substances, to name a few, exosomes are creating real excitement as a new therapeutic modality. Their production by most cell types in high concentrations also makes them attractive as biomarkers of disease.

Unfortunately, for various reasons, few exosome-based candidates have made it into clinical trials, and the majority that have are struggling with insufficient patient enrollment and funding shortages. Current developers must overcome these hurdles, as well as regulatory uncertainty and numerous manufacturing challenges, if the potential of exosomes is to be realized in the form of approved therapeutic and diagnostic products. None of the hurdles are unsurmountable, however, and given the early clinical data generated to date and the tenacity and innovativeness of biopharmaceutical researchers, the outlook remains bright.

References

  1. Cheng, Ke and Raghu Kalluir. “Guidelines for clinical translation and commercialization of extracellular vesicles and exosomes based therapeutics.” Extracellular Vesicle. 2: 100029 (2023).
  2. Shi, Hui et al. “Exosomes: Emerging Cell-Free Based Therapeutics in Dermatologic Diseases.” Cell Dev. Biol. Sec. Molecular and Cellular Pathology. 9: 14 (2021).
  3. Tan, Fei et al. “Clinical applications of stem cell-derived exosomes.” Signal Transduct. Target Ther. 9:17 (2024).
  4. Sanz-Ros, Jorge et al. “Extracellular Vesicles as Therapeutic Resources in the Clinical Environment.” J. Mol. Sci. 24: 2344 (2023).
  5. Everzom.” Accessed May 4 2024.
  6. Exosome Therapy Market- Distribution by Type of Therapy (Allogeneic and Autologous), Target Indication, Therapeutic Area, Route of Administration and Geography: Industry Trends and Global Forecasts, 2022-2040. Roots Analysis. 2022.
  7. Chavda, P. et al. “Exosome nanovesicles: A potential carrier for therapeutic delivery.” Nanotoday, 49: 101771 (2023).
  8. Sasso, Janet. “Exosome research: from platelet dust to pioneering therapeutics.” CAS Insights. 15 Dec. 2022.
  9. Balakrishnan, Sruthi S. “Exosome Therapeutics Are Paving a Path to Clinical Readiness.” BioSpace. 11 Mar. 2024.
  10. Duong, An et al. “Registered clinical trials investigating treatment with cell-derived extracellular vesicles: a scoping review.” Cytotherapy, 25 (2023).
  11. Hildreth, “The Emerging Role of Exosome Therapeutics in 2024.” Bioinformat. 9 Mar. 2024.
  12. Codiak Presents New Preclinical Data for First-in-Class Exosome Therapeutic Candidates, exoIL-12 and exoSTING, Demonstrating Potent Anti-Tumor Activity. Flagship Pioneering. 8 Nov. 2019.

Originally published on PharmasAlmanac.com on May 9, 2024.

A Media Platform for Cost-Effective and Scalable 2D and 3D Bioreactor MSC Expansion

Cell culture for mesenchymal stem (stromal) cells (MSCs) using traditional protocols and conventional media can be lengthy and require the use of large quantities of media, making it expensive and impractical for the production of cost-effective cell therapies, particularly those intended to treat large patient populations. With a media platform from RoosterBio purposely developed to provide optimal MSC expansion, it is possible to dramatically reduce both the time and cost required for MSC cell culture while generating billions of MSCs, making it possible for cell therapy companies to profitably bring their novel treatments to patients at a reasonable price.

Limitations of Typical MSC Cell Culture Process

Cell culture processes that are used today for the expansion of mesenchymal stem (or stromal) cells (MSCs) have not changed significantly since the first cell therapies were investigated. Typically, with these legacy media systems, cells are seeded at a relatively high density and then allowed to grow. With a slow cell doubling (replication) time of approximately 30 hours, it is necessary to exchange the media twice before sufficient cells are produced for harvesting and further expansion.

In addition, in order to encourage MSCs, which are adherent cells, to adequately attach to the cell culture substrate, an expensive coating often must be applied to the culture surfaces prior to seeding. In smaller vessels, the cost of the coating isn’t that noticeable, but, at large scale, it has the potential to impact the feasibility of commercialization.

Furthermore, media used for research-scale MSC cell culture cannot be used for GMP manufacture for clinical and commercial therapies. In fact, GMP-ready media may come from an entirely different supplier. Even when research and GMP media are provided by the same company, they often function differently, requiring more process development. Similarly, legacy media are designed specifically for MSC cell culture in flatware or bioreactors, and thus, when processes are transferred from 2D plasticware to 3D bioreactors, different media must be used. Each of these requires additional costly process development work.

Consequently, MSC cell culture performed in the traditional manner with legacy media is typically a lengthy, tedious, and expensive process.

A New Media Solution

Recognizing the limitations of these legacy systems and seeking a means for facilitating efficient and cost-effective MSC therapy development and manufacturing, RoosterBio developed an “MSC ecosystem” including a fit-for-purpose media platform and human MSC (hMSC) cells.

The MSC media platform offers many advantages over the legacy media that is used for MSC cell culture. Cells replicate much more quickly and can be harvested sooner without the requirement for media exchanges, reducing the time and cost for cell expansion. Higher quantities of cells are generally produced, despite the reduced process times. When the recommended processes are employed, no surface coating is necessary for efficient cell attachment, eliminating the cost of the coating and the time needed to apply it.

Figure 1: Scalable 2D MSC Processes from Bench to Pilot to Commercial

2022_Q2_Rooster_Figure 1 - Scalable 2D

Scale-up is simplified as well. The research and GMP grades of RoosterBio’s MSC media operate the same. Cell therapy developers can use lower-cost research-grade material for process development with confidence that the GMP-grade media will provide the same results for the production of clinical material without the need for further optimization (Figure 1). In addition, the media is optimized for both 2D and 3D cell culture processes (Figure 2).

Figure 2: Scalable 3D MSC Processes from Bench to Pilot to Commercial

2022_Q2_Rooster_Figure 2- Scalable 3D

Overall, RoosterBio’s media developed specifically for MSC cell culture eliminates many of the “hidden” costs associated with legacy cell culture media while providing higher yields. Avoiding media exchanges means less media consumption and waste generation. Requiring fewer passages also involves less labor and affords a reduced risk of contamination. Raw material and waste handling costs and the time to produce the cells — and therefore the cost to rent the manufacturing suite — are also reduced. As an added bonus, the cells are ultimately healthier, because they are kept in culture for a shorter period of time.

Trusting the New Process

Realizing the benefits of RoosterBio’s fit-for-purpose MSC media does require use of the recommended protocols. This is a premium media system when compared to conventional cell culture media on the market, and simply replacing that media and running the same protocols will not provide the benefits described above. It is essential for cell therapy developers to trust the RoosterBio process to reap the maximal benefits from the media.

At lab scale, legacy processes require thawing cells, plating them onto coated plates, performing two media exchanges in a row three days apart, harvesting the expanded cells, splitting them, and repeating. When using RoosterBio media, cells are thawed and plated onto uncoated plates and then harvested and split after four or five days with no second passaging step, unless a larger quantity of cells is required. Despite the shortened protocol and less time spent in the lab, the result is a higher yield of healthier cells. The cells are handled less, about half the media is consumed, and the time to product is reduced by a third or more. The result is significant time and cost savings despite the higher cost of the specially engineered media.

It’s Science, Not Magic

The fact that the media has been specifically engineered for MSC cell culture is the basis of its high performance. The same approaches, expertise, and insights used to optimize cell culture processes themselves were applied to the development of a media optimally suited for MSC cell culture.

The driver for the development of this fit-for-purpose media was the recognition that, with existing media, a tremendous quantity of media is needed to produce a sufficient number of doses of an MSC therapy, making manufacture in typical bioreactors not feasible. RoosterBio’s goal is to help MSC therapy developers advance their products through development and commercialization so they can positively affect patient lives. Solving this problem was thus crucial.

Most media suppliers aren’t willing to invest the time and resources needed to develop an optimum cell culture medium. RoosterBio did, even though such a product allowed each customer to use less media. Applying bioengineering and bioprocessing expertise led to the development of media for MSC cell culture that does more than simply not kill the MSC cells — it is designed to enable optimal cell growth. The initial focus on performance in bioreactors led to the development of media that allows for seamless transition from 2D flatware to 3D bioreactors, while the focus on clinical success led to the development of research-grade media that perform the same as GMP-grade products.

The end result: a bioengineered MSC media platform that affords much more efficient, cost-effective, and industrializable manufacturing processes that generate healthy cells — as confirmed by extensive post-expansion cell characterization.

Making the Most of the Media Platform

As with any other technology employed in the manufacture of biologic drug substances and drug products, it is recommended that the MSC media platform be adopted as early in the drug development process as possible. The later the development stage, the higher the cost to implement a change, owing to the need to demonstrate equivalency in the process, although RoosterBio is open to help clients at all development stages.

When the RoosterBio media is paired with RoosterBio MSC cells, which are well characterized and manufactured using tightly controlled processes, a certain level of expansion is guaranteed, depending on the specific process. The intent with the MSC ecosystem is to help MSC product developers with an end-to-end manufacturing solution.

In addition to the cell culture media, RoosterBio also offers upstream genetic engineering–focused media and downstream extracellular vesicle (EV) collection media. hMSCs are a clinically relevant cell source for extracellular vesicles (EVs), such as exosomes, which have been shown to be potent and able to elicit similar responses to whole hMSCs. The optimal hMSC manufacturing process enables hMSCs to be turned into an optimal EV manufacturing cell, because MSCs are the cell type with the most highest therapeutic relevancy for exosome production. Consequently, the RoosterBio MSC media platform has applications beyond MSC expansion and could be the foundation for the industrialization of exosome generation.

Additionally, while RoosterBio remains focused on MSCs, the concept should work equally well with other types of cells, even if they have different requirements, including induced pluripotent stem cells (iPSCs), T cells, and so on.

A Quantified Value Proposition

MSCs as cell therapies and a source of other drug substances, such as EVs, are a disruptive technology and, consequently, will have a fairly extensive life cycle. As the market expands, the demand for cost-effective, efficient, and high-yielding MSC culture processes will grow exponentially.

The RoosterBio MSC platform, including both specially designed cells and media, allows MSC cell therapy developers to invest their intellectual capital on formulation development and clinical studies, rather than on the raw materials and the manufacturing process.

The fit-for-purpose media, regardless of whether used with RoosterBio MSCs or others, allows for reduced resource consumption and waste generation during the production of higher quantities of healthy cells in much less time, all of which adds up to a lower cost per cell. It also enables seamless scale-up from research to commercial GMP production, including the transfer of processes from 2D flatware to 3D bioreactors.

Figure 3. Comparative Results for the RoosterBio Cell-Culture System

2022_Q2_Rooster_Figure 3- Comparative Results

Comparative studies performed for 2D and 3D culture process using the RoosterBio media and protocol and various commercially available conventional media and corresponding protocols clearly demonstrate the benefits of the RoosterBio media platform (Figure 3). No other media on the market offers such a quantified value proposition.

Originally published on PharmasAlmanac.com on April 21, 2022

Moving Beyond the Industrialization of MSCs

Tim Kelly, the new Chief Executive Officer at RoosterBio, met with Pharma’s Almanac’s Scientific Editor in Chief to discuss Rooster’s standardized stem cell product platforms that enable rapid clinical and commercial translation and its commitment to industrializing regenerative medicine supply chains to accelerate innovation.

David Alvaro (DA): Tim, can you share a bit about your professional history and the transition to your current role as CEO of RoosterBio?

Tim Kelly (TK): I am a biochemist by training and have a Ph.D. in molecular genetics and biochemistry. After completing my studies, I went straight into industry, joining a contract development and manufacturing organization (CDMO) that eventually became Fujifilm Diosynth Biotechnologies. I was there for about six years in a quality control function and during that time the facility transitioned from clinical to commercial, with two licensed biologics. Next, I worked at the CDMO KBI Biopharma for 15 years and participated in its significant growth, including launching a cell therapy CDMO business focused on autologous immunotherapy applications.

After that, I worked at the gene therapy company Asklepios Biopharmaceutical (AskBio) to launch their manufacturing business unit. I had previously met Jon Rowley (RoosterBio’s founder) and also had relationships with some of Rooster’s Series B investors at Dynamk Capital. As a result, I was aware of the company’s history and growth opportunities. It is rewarding to again be in a position to help a company achieve that transition to where it can really have a commercial impact on drug development and manufacturing, and it’s going to be a very exciting next several years for us at RoosterBio. 

DA: What is your vision for RoosterBio?

TK: Cell and gene therapies are the most rapidly growing area of our industry, and they bring unbelievable promise and opportunity, but also a really unique set of challenges, particularly on the CMC and manufacturing side. That’s really where I’ve spent 25 years of my career: solving development and manufacturing challenges for biologics. So, this is where my background and RoosterBio’s value proposition really intersect, and we hope to amplify these areas over the next 12–24 months..

The team at RoosterBio has clearly done a tremendous job getting these products developed, commercialized and into the hands of customers. But what we want to do in the years to come is to extend the value proposition from a complete product solution to a complete systems solution that incorporates the development and manufacture of our customers’ products –– including both the mesenchymal stromal/stem cells (MSCs) therapeutics, as well as next-generation MSC products, such as extracellular vesicles (EVs) and exosomes, which are really exciting elements of MSC therapy. Additionally, we are building solutions to enable the genetic modification of MSCs during GMP manufacturing to forward-engineer cells that produce specific targets that can then be potentially incorporated into those EVs and provide other unique therapeutic opportunities.

The key is to establish a position of technology leadership. We were able to do that in the AAV space at AskBio and in analytical characterization and formulation development at KBI. RoosterBio has already done that for MSCs. Now, we are poised to expand on that technology leadership in terms of our development services capabilities and our manufacturing solutions toolbox, which could include building our own GMP manufacturing capacity. In the near term we will leverage partners in the space that are  well-aligned with us and for whom we know  will be able to deliver successful solutions.

So overall, my hopes and plans for RoosterBio fall into two areas. The first is expanding the services business so that we can provide end-to-end support beyond the cellular starting materials and bioprocess media to include complete solutions that help our customers get into the clinic and get their products to patients. The second is to facilitate end-to-end solutions for next-generation MSC technologies and products.

DA: Have any aspects of that vision and strategy changed since you assumed the helm of RoosterBio?

TK: Rooster has certainly been on that path toward next-generation MSC products and applications. In fact, Rooster customers have been using our MSCs for genetic engineering and to make EVs for years. Perhaps the difference I bring to the business is my experience in CMC manufacturing and my ability to operationalize Rooster’s development and manufacturing strategy. I have built this part of several businesses in the past and feel like I have a pretty good sense of who we need, what we need, and how to approach customers and effectively partner with them to deliver successes in process development and manufacturing services.

I bring a focus on building the CMC and contract services element as a way to introduce our products to more customers and fundamentally to ensure that our customers are going to be successful with our products. We don’t want them to buy our cells and media and then run off and try and figure it out themselves. We want to enable them to be successful and to really leverage the unique power of our toolbox to accelerate their path to the clinic.

DA: What does Rooster really mean when you discuss industrializing the regenerative medicine supply chain? Why is that so important, and what are the benefits to your customers and beyond?

TK: It isn’t just about having GMP-grade materials available; there is much more to it. RoosterBio has redesigned the way that MSC-based products can be produced by establishing standardized, off-the-shelf, and GMP-certified cell banks derived from multiple donors paired with GMP-grade media. This has been achieved through upfront tissue sourcing, designing GMP-grade materials suitable for scalable manufacturing, and optimizing a process for the rapid expansion of MSCs. Altogether, these efforts have made it possible to get the right cells, in the right manufacturing-friendly formats, at the right quality grade, and available “off-the-shelf”; which accelerates product development, innovation, and the rapid transition from development to cGMP manufacturing.

Layered on top are the regulatory advantages that come with having drug master files (DMFs) in place for our MSC and media products. Most of the CMC section for an MSC-based product involves the tissue source, the manufacture and thorough safety testing of the cell banks, as well as the media. If a customer chooses to partner with RoosterBio, they already have 70% of their CMC section completed simply by cross-referencing the DMF, which shaves multiple years off of their development timelines and many millions of dollars.

In addition, having development-grade materials in manufacturing-friendly formats that are aligned with our GMP-grade products is  key to the industrialization of MSCs. Research materials are affordable and accessible, and the transition from research into development and manufacturing is seamless. By enabling applied research, we are contributing to the development of novel medicines that will help patients, which is the ultimate goal.

The analogy our team has used is to think of MSCs like microprocessors in computers. If you want to bring a new high-tech product to market and you start by designing and building your own microprocessor, it will take many years and millions of dollars just to do that part. If you partner with Intel or a similar company and leverage existing standardized components, you get your technology product on the market years faster than you otherwise would have — and with best-in-class technology. That allows you to focus on the value proposition of your product and less about re-inventing a supply chain that already exists elsewhere. We are essentially standardizing and industrializing what is currently a highly fragmented supply chain for the cell therapy market, certainly the MSC-focused part of the industry.

In one recent example, a synthetic biology company developing a COVID-19 treatment was able to take a modified MSC from proof-of-concept to an approved IND in less than six months. Of course, the biology and timing can vary depending on the clinical specifics. But even for a COVID-19 application, it would not have been possible to move that quickly without being able to leverage the DMF and GMP-ready materials provided by RoosterBio. That example is somewhat unique, considering the COVID-19 regulatory landscape, but it does illustrate what is possible in terms of being able to accelerate these products by multiple years and reduce the overall cost to bring MSC products to market. 

DA: What can you tell me about the different types of customers you support and the different needs they have?

TK: One of the nice things about our business model is that there is a hyper-focus on customers. If customers only want research-grade cells and media, we make them readily available and affordable. We have MSC1.0 customers that are using native cells essentially as we have banked them to produce products used in different types of tissue repair and regeneration applications, such as traumatic brain injury, acute kidney injury, macular damage, etc. There are at least 10 MSC1.0 products approved globally — with several close to approval in the United States. 

Our next-gen MSC2.0 customers are developing advanced MSC products, with many of them focused on using native MSCs in combination with medical devices or as subcomponents of engineered tissues or organs. Our MSC banks are also being expanded and used as a source of EVs/exosomes, and these MSC2.0 products are being used for cardiac, cancer, pulmonary, and other therapeutic areas with significant unmet need.

In my view, some of the most exciting work is being performed by customers that are genetically modifying our MSCs and then deriving EVs from them for use in a broad range of therapeutic areas, including oncology and many other diseases. These products are not primary cells, which is important, because primary cells can be challenging, not only with respect to regulatory considerations but also with regard to concerns about how they may behave once administered — such as, for example, whether they will become cancerous. Having all of the power of the cell in a non-cell vesicle that has been modified for targeted delivery is really powerful.  

DA: When you think about their full potential, how generalizable are MSCs and MSC-based therapies with respect to the therapeutic areas they can impact?

TK: I think their potential is really broad. Once you get into the area of genetic engineering, you can target lots of different applications outside of regenerative medicine. Even with MSC 1.0, which really involves regenerative medicine applications, there are many interesting projects, including several looking at how MSC-derived products could be used to address different aging considerations. Bioprinting of artificial tissues is also an exciting area with lots of opportunity and the potential to address a huge unmet need. MSCs are already being investigated in almost all therapeutic areas and will be a big part of how regenerative medicines get developed.

DA: Can you tell me a little bit about the challenges in reaching out to customers that don’t realize how your MSCs can accelerate their projects and how you demonstrate their value?

TK: There are a lot of people in the cell therapy and regenerative medicine fields that are not working with MSCs. They may be working with induced pluripotent stems cells (iPSCs), other types of stem cells, T cells, NK cells, and more. It is not always easy, given the biases that folks have in terms of what MSCs can potentially bring to a project.

What translates the most is the fact that there is no other cell type that has an industrialized supply chain. In fact, with iPSCs (induced pluripotent stem cells), there are some unique challenges or barriers to the effort to establish an industrialized supply chain. Often, when people see what RoosterBio has done in terms of our products and processes and how those combined can accelerate clinical development, that really resonates.

Things definitely can depend on the customer and the application. In some cases, there might be an opportunity, perhaps through genetic engineering, to create an MSC product that could address that same need. In other cases, MSCs simply aren’t the answer.

DA: Are there any other cell types that RoosterBio might try to industrialize in an analogous way?

TK: We definitely get lots of questions about our interest in expanding into other cell types. As we observe how the industry is developing, we definitely see opportunity in applying the RoosterBio business model to other areas of the advanced therapies supply chain.  We are interested, but the question that must be addressed is how to prioritize that type of work given everything else that RoosterBio is currently trying to do in the business.

Expanding into other cell types or technology areas, however, does present opportunities for us in the years to come — trying to apply our business model to other cell types and achieve a similar type of value in the cells that have both similar and different applications to MSCs.

The most obvious candidate is iPSCs, because there is huge interest in that space. Although there is much more clinical development occurring with MSCs in terms of the numbers of INDs and clinical trials, iPSCs have different potential applications, because MSCs have fixed differentiation capabilities relative to iPSCs. We might perhaps consider partnering with a company with iPSC expertise to apply what we’ve done with in MSCs to the iPSC space. Other types of stem cells, such as embryonic stem cells, also present unique opportunities, but iPSCs are the obvious first choice.

It is worth mentioning at this point that there are different source tissues in the MSC space itself. Historically, RoosterBio has mainly worked with bone marrow, but we do offer umbilical cord–derived stem cells as a research-grade product. We are currently working to introduce GMP-grade umbilical cord–derived MSC working cell banks. Additional work is also underway on adipose-derived MSCs. The goal is to expanding the range of products so that customers with specific applications can choose MSC products with the optimum properties. 

DA: To what extent is there a clear regulatory path and strong guidance for MSC 1.0 and 2.0 developers, and how crucial is that CMC focus?

TK: It is still important for both applications. To date, no robust MSC-based therapeutics have been approved by the U.S. Food and Drug Administration, but they are on the market in Europe and Asia and other parts of the world. Even in the 1.0 applications, having that really well-thought-out and comprehensive quality and regulatory package is critical. I expect that we will soon see approvals of MSC products in the United States, and the fact that none have occurred yet contributes to skepticism in some quarters about the effectiveness of MSC-based approaches.

In terms of the 2.0 applications, the regulatory pathway for EVs is actually simpler, because the living cell component is removed. Whether scientifically based or not, there are concerns about administering primary cells outside of autologous treatments, which have become reasonably well accepted from a safety point of view, because each patient’s cells are manipulated and then returned to that same patient. But administering cells as therapeutics poses different types of challenges than administering a protein, small molecule, or even a viral vector, which of course have their own considerations. I hope that, as EVs continue to advance, they will enable a new wave of MSC-related therapies, because when you take the cell out of the equation, you have a product that is easier to characterize, store, and administer. As a result, some of the regulatory considerations are taken off the table.

Regardless, there will definitely be challenges with respect to setting appropriate specifications for purity, quality, identity, potency, and so forth. These are some of the challenges that we’re working hard at RoosterBio to solve.

For instance, our MSC research products and cGMP cell banks are better characterized than any other MSC available on the market today. We do more to characterize and understand our cells and ultimately our EVs than any other suppliers, because we know that a higher level of knowledge is essential to getting these products through the regulatory hurdles. There has been some latitude given to next-generation therapies targeting orphan indications with significant unmet need with respect to providing limited characterization data, because manufacturers have claimed that these products are very difficult to characterize, and only so much can be done.

The truth is that we can do more, and companies are choosing not to. That is not the case with RoosterBio; we are pushing the envelope in that space because we foresee that the FDA will ultimately increase its expectations to the point where cells and vectors need to be characterized as well as monoclonal antibodies. The faster we can get there, the more products we will get approved and the more patients we will be able to treat and help.

DA: As you look to the future, what changes do you anticipate or hope to see in the MSC space?

TK: The MSC market is rapidly developing, and we expect it will pass through some inflexion points to eventually reach the level of exuberance now seen for CAR-T and related therapies. A lot of the tools that have been developed by RoosterBio and other players within the industry are going to fuel that, including genetic engineering capabilities.

All of the pieces are starting to come together: The MSC cell type has a long-standing safety profile. RoosterBio has developed a bioprocess platform that provides product developers with a nearly unlimited and relatively inexpensive supply of MSCs. And now, the genetic engineering toolbox allows developers to weaponize these cells to maximize their therapeutic potential.

As a result, we expect to see companies exploiting many of the innate capabilities or characteristics of MSCs, from longevity to anti-inflammatory properties, to even using them as drug delivery devices — whether as the cells themselves or in the form of EVs that the cells secrete. We also expect to see companies using MSCs or EVs to transfer their properties to other cells and to prime those other cells with their characteristics, such as in the form of adoptive therapies.

Overall, we believe the MSC market is going to explode as a result of standardized, off-the-shelf tools and as a result of MSCs’ innate therapeutic activity. Those two factors coming together is really going to lead to the rapid increase in the number of MSCs being investigated for therapeutic applications, and eventually an acceleration of MSC products on the market.

A critical milestone for MSCs will be that first FDA approval of an MSC product, which we are confident will happen in the very near term. And it will be followed by approval and commercialization of EV-based products, which will help demonstrate the clinical efficacy of MSCs out in the market, which will be another huge inflection point.

The last stage will be approval of completely unique, genetically engineered MSCs and EVs that deliver clinical efficacy that no one thought possible with MSC-based products and which will require determining how to put everything together from the CMC, regulatory, and clinical points of view. That will be a real watershed moment for the MSC space, when all the advantages of using MSCs will be recognized.

Of course, there is skepticism, but that will be overcome. In fact, once those genetically modified products ultimately become successful in the clinic and get commercialized, they could serve as safer nonviral alternatives — as cells or EVs — to viral vectors for the delivery of genetic payloads. 

And at the same time as these developments are occurring, the bioprinting of artificial tissues and organs will continue to progress, and that whole area provides another massive opportunity for MSCs and other cell types due to the sheer volume of cells required for ex vivo tissue engineering.

DA: For RoosterBio in particular, are there any new products or service offerings that you will be introducing in the near term?

TK: From a product point of view, we are actively developing expanded portfolio products with a focus on EVs and genetically engineered MCSs. We are also continuing to optimize the products that we already offer, as well as developing GMP-compliant versions of products currently only available as research-grade materials.

RoosterBio has so far largely focused on products for upstream production — growing the cells, getting the cells to secrete EVs, or genetically modifying the cells. Going forward, we will be dedicating some of our work to downstream development, particularly with respect to EV-based products and the development of bioprocess materials that facilitate EV purification and formulation.

With respect to services, we are working to complete the entire value chain for EVs through downstream processing and formulation and analytical characterization, particularly pushing the envelope on the analytics piece and developing more genetic engineering tools. We currently have a product that facilitates highly efficient lentiviral transduction of our MSCs, which we want to expand to include adeno-associated virus (AAV) and also nonviral means of introducing DNA, such as electroporation and transfection. The goal is to have a toolbox so that customers have access to ready-to-go solutions however they intend to develop their products.

Our ultimate aspiration is to meet all of our customers’ needs regardless of the development approaches they are taking and to provide the products, services, and bioprocess solutions that will help them reach the clinic and the market faster than they could otherwise, to ultimately get these innovative therapies to the patients that need them.

Originally published on PharmasAlmanac.com on March 12, 2022